Generation and profiling of fully human HuCAL GOLD-derived therapeutic antibodies specific for human CD38

ABSTRACT

The present invention provides novel antibodies and methods for using recombinant antigen-binding regions and antibodies and functional fragments containing such antigen-binding regions that are specific for CD38, which plays an integral role in various disorders or conditions. These methods take advantage of newly discovered antibodies and surprising properties of such antibodies, such as the ability to bind CD38 of minipig origin and the ability to induce, by cross-linking, specific killing of cells that express CD38. These antibodies as well as the novel methods for using those antibodies can be used to treat, for example, hematological malignancies such as multiple myeloma.

SUMMARY OF THE INVENTION

The present invention relates to an isolated antigen-binding region thatis specific for CD38, which comprises (i) an H-CDR3 region depicted inSEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106 or(ii) an H-CDR3 region that has at least a sixty percent identity to anH-CDR3 region depicted in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105 or 106

The present invention furthermore relates to an isolated antibody orfunctional fragment thereof that is specific for CD38, which comprises(i) a variable heavy chain depicted in SEQ ID NO: 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105 or 106 or (ii) a variable heavy chain thathas at least a sixty percent identity to a variable heavy chain depictedin SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106.

Additionally, the present invention relates to an isolatedantigen-binding region that is specific for CD38, which comprises (i) anL-CDR3 region depicted in SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 109 or 110 or (ii) an L-CDR3 region that has atleast a sixty percent identity to an L-CDR3 region depicted in SEQ IDNO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 109 or110.

Also, the present invention relates to an isolated antibody orfunctional fragment thereof, which comprises (i) a variable light chaindepicted in SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 109 or 110 or (ii) a variable light chain that has at leasta sixty percent identity to a variable light chain depicted in SEQ IDNO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 109 or110.

The present invention further relates to a variable heavy chain of anisolated antigen-binding region that is encoded by (i) a nucleic acidsequence comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91or (ii) a nucleic acid sequences that hybridizes under high stringencyconditions to the complementary strand of SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90 or 91, wherein said antigen-binding region is specificfor CD38.

The present invention also relates to a variable light chain of anisolated antigen-binding region that is encoded by (i) a nucleic acidsequence comprising SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 107 or 108 or (ii) a nucleic acid sequences thathybridizes under high stringency conditions to the complementary strandof SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 107 or 108, wherein said antibody or functional fragment thereof isspecific for CD38.

Further, the present invention relates to an isolated nucleic acidsequence that encodes an antigen-binding region of a human antibody orfunctional fragment thereof that is specific for CD38.

Additionally, the invention relates to a nucleic acid sequence encodinga variable heavy chain of an isolated antigen-binding region, whichcomprises (i) a sequence selected from the group consisting of SEQ IDNOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90 and 91 or (ii) a nucleic acidsequence that hybridizes under high stringency conditions to thecomplementary strand of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90or 91, wherein said antigen-binding region is specific for CD38.

The present invention also relates to a nucleic acid sequence encoding avariable light chain of an isolated antigen-binding region, whichcomprises (i) a sequence selected from the group consisting of SEQ IDNOS: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 107 and108 or (ii) a nucleic acid sequence that hybridizes under highstringency conditions to the complementary strand of SEQ ID NO: 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 107 or 108 whereinsaid antigen-binding region is specific for CD38.

The present invention further relates to a method of inducing specifickilling of tumor cells that express CD38, wherein said specific killingoccurs by CD38 cross-linking, comprising the step of incubating saidcells in the presence of a sufficient amount of an isolated human orhumanized anti-CD38 antibody or a functional fragment thereof, whereinsaid human or humanized anti-CD38 antibody comprises (i) a nucleic acidsequence encoding a heavy chain depicted in SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90 or 91 or (ii) a nucleic acid sequences that hybridizesunder high stringency conditions to the complementary strand of SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91, wherein said antibody or afunctional fragment thereof is specific for CD38.

Additionally, the present invention relates toA method of inducingspecific killing of tumor cells that express CD38, wherein said specifickilling occurs by CD38 cross-linking, comprising the step of incubatingsaid cells in the presence of a sufficient amount of an isolated humanor humanized anti-CD38 antibody or a functional fragment thereof,wherein said human or humanized anti-CD38 antibody comprises (i) anucleic acid sequence encoding a light chain depicted in SEQ ID NO: 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 107 or 108 or(ii) a nucleic acid sequences that hybridizes under high stringencyconditions to the complementary strand of SEQ ID NO: 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 107 or 108, wherein saidantibody or a functional fragment thereof is specific for CD38.

Also, the present invention relates to a method of inducing specifickilling of tumor cells that express CD38, wherein said specific killingoccurs by CD38 cross-linking, comprising the step of incubating saidcells in the presence of a sufficient amount of an isolated human orhumanized anti-CD38 antibody or a functional fragment thereof, whereinsaid human or humanized anti-CD38 antibody or said functional fragmentthereof comprises (i) a heavy chain amino acid sequence depicted in SEQID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106 or (ii)a variable heavy chain that has at least a sixty percent identity to avariable heavy chain depicted in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105 or 106.

Also, the present invention relates to a method of inducing specifickilling of tumor cells that express CD38, wherein said specific killingoccurs by CD38 cross-linking, comprising the step of incubating saidcells in the presence of a sufficient amount of an isolated human orhumanized anti-CD38 antibody or a functional fragment thereof, whereinsaid human or humanized anti-CD38 antibody comprises (i) and/or a lightchain amino acid sequence depicted in SEQ ID NO: 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 109 or 110 or (ii) a variable lightchain that has at least a sixty percent identity to a variable lightchain depicted in SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 109 or 110.

Furthermore, the present invention relates to a method of detectingspecific killing of tumor cells that express CD38, by CD38cross-linking, comprising the steps of:

-   -   (i) administering to a subject in need thereof an effective        amount of a human or humanized anti-CD38 antibody or a        functional fragment thereof, and    -   (ii) detecting the specific killing activity of said human or        humanized anti-CD38 antibody or said functional fragment        thereof.

Also, the present invention relates to a method of detecting thepresence of CD38 in a tissue or a cell of minipig origin, comprising thesteps of:

-   -   (i) allowing a human or humanized anti-CD38 antibody or a        functional fragment thereof to come into contact with said CD38,        and    -   (ii) detecting the specific binding of said human or humanized        anti-CD38 antibody or functional fragment thereof to said CD38        minipig cells, wherein said antibody or functional fragment        thereof is also able to specifically bind to CD38 of human        origin.

Furthermore, the present invention relates to A method of detecting CD38in a CD38-expressing erythrocyte, comprising the steps of

-   -   (i) allowing a human or humanized anti-CD38 antibody or a        functional fragment thereof to come into contact with said        CD38-expressing erythrocyte, and    -   (ii) detecting the specific binding of said human or humanized        anti-CD38 antibody or functional fragment thereof to said        CD38-expressing erythrocytes, wherein said antibody or        functional fragment thereof is also able to specifically bind to        human CD38 from a cell or tissue other than human erythrocytes.

The present invention also relates to an isolated antibody or functionalfragment thereof according to the present invention, which comprises (i)an H-CDR3 region depicted in SEQ ID NO: 21 or 22 or (ii) an H-CDR3region at least a sixty percent identity thereto, and that is specificfor human CD38 and marmoset CD38.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a provides nucleic acid sequences of various antibody variableheavy regions for use in the present invention.

FIG. 1b provides amino acid sequences of various antibody variable heavyregions for use in the present invention. CDR regions HCDR1, HCDR2 andHCDR3 are designated from N- to C-terminus in boldface.

FIG. 2a provides nucleic acid sequences of various antibody variablelight regions for use in the present invention.

FIG. 2b provides amino acid sequences of various antibody variable lightregions for use in the present invention. CDR regions LCDR1, LCDR2 andLCDR3 are designated from N- to C-terminus in boldface.

FIG. 3 provides amino acid sequences of variable heavy regions ofvarious consensus-based HuCAL antibody master gene sequences. CDRregions HCDR1, HCDR2 and HCDR3 are designated from N- to C-terminus inboldface.

FIG. 4 provides amino acid sequences of variable light regions ofvarious consensus-based HuCAL antibody master gene sequences. CDRregions LCDR1, LCDR2 and LCDR3 are designated from N- to C-terminus inboldface.

FIG. 5 provides the amino acid sequence of CD38 (SWISS-PROT primaryaccession number P28907).

FIG. 6 provides the nucleotide sequences of the heavy and light chainsof chimeric OKT10.

FIG. 7 provides the DNA sequence of pMORPH®_h_IgG1_1 (bp 601-2100) (SEQID NO: 74): The vector is based on the pcDNA3.1+ vectors (Invitrogen).The amino acid sequence of the VH-stuffer sequence is indicated in bold,whereas the final reading frames of the VH-leader sequence and theconstant region gene are printed in non-bold. Restriction sites areindicated above the sequence. The priming sites of the sequencingprimers are underlined.

FIG. 8 provides the DNA sequence of Ig kappa light chain expressionvector pMORPH®_h_Igκ_1 (bp 601-1400) (SEQ ID NO: 75): The vector isbased on the pcDNA3.1+ vectors (Invitrogen). The amino acid sequences ofthe Vκ-stuffer sequence is indicated in bold, whereas the final readingframes of the Vκ-leader sequence and of the constant region gene areprinted in non-bold. Restriction sites are indicated above the sequence.The priming sites of the sequencing primers are underlined.

FIG. 9 provides the DNA sequence of HuCAL Ig lambda light chain vectorpMORPH®_h_Igλ_1 (bp 601-1400) (SEQ ID NO: 76): The amino acid sequenceof the V

-stuffer sequence is indicated in bold, whereas the final reading framesof the V

-leader sequence and of the constant region gene are printed innon-bold. Restriction sites are indicated above the sequence. Thepriming sites of the sequencing primers are underlined.

FIG. 10 provides different combinations of heavy and light chains in theFab/IgG format for use in the present invention

FIG. 11 provides CD38-expression analysis of Lymphocytes andErythrocytes obtained by FACS. PBMCs and Erythrocytes were isolated fromwhole blood of cynomolgus, rhesus and marmoset by density gradientcentrifugation followed by FACS-analysis using anti-CD38 Fab antibodiesMOR03087 (A, right histograms, light arrow) and MOR03088 (B, righthistograms; light arrow). An irrelevant Fab-antibody (A & B, lefthistograms; black arrow) was used as a negative control.

FIG. 12 provides CD38 expression analysis of Lymphocytes andErythrocytes obtained by FACS.

PBMCs and Erythrocytes were isolated from whole blood of human,cynomolgus and marmoset by density gradient centrifugation followed byFACS-analysis using anti-CD38 IgG1 MOR03087 (right histograms; whitearrow). An irrelevant IgG1 control antibody (A & B, left histograms;black arrow) was used as a negative control.

FIG. 13 provides a comparative overview of Cross-Reactivity of differentanti-CD38 antibodies.

FIGS. 14(a) and 14(b) delineate the CDR and FR regions for certainantibodies for use in the invention and compare amino acids at agivenposition to each other and to corresponding consensus sequences.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novel antibodies andmethods of using antibodies that are specific to or have a high affinityfor CD38 and can deliver a therapeutic benefit to a subject. Theantibodies, which may be human or humanized, can be used in manycontexts, which are more fully described herein. Suitable antibodies foruse in the present invention are disclosed in U.S. 60/614,471, whichhereby is incorporated by reference.

A “human” antibody or functional human antibody fragment is herebydefined as one that is not chimeric (e.g., not “humanized”) and not from(either in whole or in part) a non-human species. A human antibody orfunctional antibody fragment can be derived from a human or can be asynthetic human antibody. A “synthetic human antibody” is defined hereinas an antibody having a sequence derived, in whole or in part, in silicofrom synthetic sequences that are based on the analysis of known humanantibody sequences. In silico design of a human antibody sequence orfragment thereof can be achieved, for example, by analyzing a databaseof human antibody or antibody fragment sequences and devising apolypeptide sequence utilizing the data obtained therefrom. Anotherexample of a human antibody or functional antibody fragment, is one thatis encoded by a nucleic acid isolated from a library of antibodysequences of human origin (i.e., such library being based on antibodiestaken from a human natural source).

A “humanized antibody” or functional humanized antibody fragment isdefined herein as one that is (i) derived from a non-human source (e.g.,a transgenic mouse which bears a heterologous immune system), whichantibody is based on a human germline sequence; or (ii) chimeric,wherein the variable domain is derived from a non-human origin and theconstant domain is derived from a human origin or (iii) CDR-grafted,wherein the CDRs of the variable domain are from a non-human origin,while one or more frameworks of the variable domain are of human originand the constant domain (if any) is of human origin.

As used herein, an antibody “binds specifically to,” is “specificto/for” or “specifically recognizes” an antigen (here, CD38) if suchantibody is able to discriminate between such antigen and one or morereference antigen(s), since binding specificity is not an absolute, buta relative property. In its most general form (and when no definedreference is mentioned), “specific binding” is referring to the abilityof the antibody to discriminate between the antigen of interest and anunrelated antigen, as determined, for example, in accordance with one ofthe following methods. Such methods comprise, but are not limited toWestern blots, ELISA-, RIA-, ECL-, IRMA-tests, FACS, IHC and peptidescans. For example, a standard ELISA assay can be carried out. Thescoring may be carried out by standard color development (e.g. secondaryantibody with horseradish peroxide and tetramethyl benzidine withhydrogenperoxide). The reaction in certain wells is scored by theoptical density, for example, at 450 nm. Typical background (=negativereaction) may be 0.1 OD; typical positive reaction may be 1 OD. Thismeans the difference positive/negative can be more than 10-fold.Typically, determination of binding specificity is performed by usingnot a single reference antigen, but a set of about three to fiveunrelated antigens, such as milk powder, BSA, transferrin or the like.It is possible for an antibody to be “specific to” or “specific for” anantigen of 2 or more cells/tissues and/or 2 or more species, providedthat the antibody meets binding criteria for each of such cells/tissuesand species, for example. Accordingly, an antibody may bind specificallyto the target antigen CD38 on various cell types and/or tissues, e.g.erythrocytes, lymphocytes isolated from peripheral blood, spleen orlymph-nodes. In addition, an antibody may be specific to both CD38 ofone species and CD38 of another species.

“Specific binding” also may refer to the ability of an antibody todiscriminate between the target antigen and one or more closely relatedantigen(s), which are used as reference points, e.g. between CD38 andCD157. Additionally, “specific binding” may relate to the ability of anantibody to discriminate between different parts of its target antigen,e.g. different domains or regions of CD38, such as epitopes in theN-terminal or in the C-terminal region of CD38, or between one or morekey amino acid residues or stretches of amino acid residues of CD38.Also, as used herein, an “immunoglobulin” (Ig) hereby is defined as aprotein belonging to the class IgG, IgM, IgE, IgA, or IgD (or anysubclass thereof), and includes all conventionally known antibodies andfunctional fragments thereof. A “functional fragment” of anantibody/immunoglobulin hereby is defined as a fragment of anantibody/immunoglobulin (e.g., a variable region of an IgG) that retainsthe antigen-binding region. An “antigen-binding region” of an antibodytypically is found in one or more hypervariable region(s) of anantibody, i.e., the CDR-1, -2, and/or −3 regions; however, the variable“framework” regions can also play an important role in antigen binding,such as by providing a scaffold for the CDRs. Preferably, the“antigen-binding region” comprises at least amino acid residues 4 to 103of the variable light (VL) chain and 5 to 109 of the variable heavy (VH)chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111of VH, and particularly preferred are the complete VL and VH chains(amino acid positions 1 to 109 of VL and 1 to 113 of VH; numberingaccording to WO 97/08320). A preferred class of immunoglobulins for usein the present invention is IgG. “Functional fragments” of the inventioninclude the domain of a F(ab′)₂ fragment, a Fab fragment and scFv. TheF(ab′)₂ or Fab may be engineered to minimize or completely remove theintermolecular disulphide interactions that occur between the C_(H1) andC_(L) domains.

The term “parental binder” as used in connection with the presentinvention denotes any binder which has not undergone the process ofoptimization. A process of optimization is described elsewhere in thepresent specification.

The term “binder” as used in connection with the present invention maybe used in a synonymous manner as the term “immunoglobulin” or“antibody”.

An antibody for use in the invention may be derived from a recombinantantibody library that is based on amino acid sequences that have beendesigned in silico and encoded by nucleic acids that are syntheticallycreated. In silico design of an antibody sequence is achieved, forexample, by analyzing a database of human sequences and devising apolypeptide sequence utilizing the data obtained therefrom. Methods fordesigning and obtaining in silico-created sequences are described, forexample, in Knappik et al., J. Mol. Biol. (2000) 296:57; Krebs et al.,J. Immunol. Methods. (2001) 254:67; and U.S. Pat. No. 6,300,064 issuedto Knappik et al., which hereby are incorporated by reference in theirentirety.

Antibodies for Use in the Invention Throughout this document, referenceis made to the following representative antibodies for use in theinvention: “antibody nos.” or “LACS” or “MOR” 3076 or 03076, 3078 or03078, 3081 or 03081, 3085 or 03085, 3086 or 03086, 3087 or 03087, 3088or 03088, 3089 or 03089, 3101 or 03101, 3102 or 03102, 3127 or 03127,3128 or 03128, 3129 or 03129, 3130 or 03130, 3131 or 03131, 6183 or06183, 6184 or 06184, 6185 or 06185, 6186 or 06186, 6187 or 06187, 6188or 06188, 6189 or 06189, 6190 or 06190, 6192 or 06192, 6195 or 06195,6197 or 06197, 6200 or 06200, 6201 or 06201, 6204 or 06204, 6214 or06214, 6278 or 06278, 6279 or 06279. LAC 3076 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 1 (DNA)/SEQID NO: 16 (protein) and a variable light region corresponding to SEQ IDNO: 31 (DNA)/SEQ ID NO: 46 (protein). LAC 3078 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 2 (DNA)/SEQID NO: 17 (protein) and a variable light region corresponding to SEQ IDNO: 32 (DNA)/SEQ ID NO: 47 (protein). LAC 3081 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 3 (DNA)/SEQID NO: 18 (protein) and a variable light region corresponding to SEQ IDNO: 33 (DNA)/SEQ ID NO: 48 (protein). LAC 3085 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 4 (DNA)/SEQID NO: 19 (protein) and a variable light region corresponding to SEQ IDNO: 34 (DNA)/SEQ ID NO: 49 (protein). LAC 3086 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 5 (DNA)/SEQID NO: 20 (protein) and a variable light region corresponding to SEQ IDNO: 35 (DNA)/SEQ ID NO: 50 (protein). LAC 3087 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 6 (DNA)/SEQID NO: 21 (protein) and a variable light region corresponding to SEQ IDNO: 36 (DNA)/SEQ ID NO: 51 (protein). LAC 3088 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 7 (DNA)/SEQID NO: 22 (protein) and a variable light region corresponding to SEQ IDNO: 37 (DNA)/SEQ ID NO: 52 (protein). LAC 3089 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 8 (DNA)/SEQID NO: 23 (protein) and a variable light region corresponding to SEQ IDNO: 38 (DNA)/SEQ ID NO: 53 (protein). LAC 3101 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 9 (DNA)/SEQID NO: 24 (protein) and a variable light region corresponding to SEQ IDNO: 39 (DNA)/SEQ ID NO: 54 (protein). LAC 3102 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 10 (DNA)/SEQID NO: 25 (protein) and a variable light region corresponding to SEQ IDNO: 40 (DNA)/SEQ ID NO: 55 (protein). LAC 3127 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 11 (DNA)/SEQID NO: 26 (protein) and a variable light region corresponding to SEQ IDNO: 41 (DNA)/SEQ ID NO: 56 (protein). LAC 3128 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 12 (DNA)/SEQID NO: 27 (protein) and a variable light region corresponding to SEQ IDNO: 42 (DNA)/SEQ ID NO: 57 (protein). LAC 3129 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 13 (DNA)/SEQID NO: 28 (protein) and a variable light region corresponding to SEQ IDNO: 43 (DNA)/SEQ ID NO: 58 (protein). LAC 3130 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 14 (DNA)/SEQID NO: 29 (protein) and a variable light region corresponding to SEQ IDNO: 44 (DNA)/SEQ ID NO: 59 (protein). LAC 3131 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 15 (DNA)/SEQID NO: 30 (protein) and a variable light region corresponding to SEQ IDNO: 45 (DNA)/SEQ ID NO: 60 (protein). Furthermore, optimized clones,which were derived from the parental binders MOR03087 and MOR03088,comprise the following: MOR06183 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 77 (DNA)/SEQ ID NO: 92(protein). MOR06184 represents an antibody having a variable heavyregion corresponding to SEQ ID NO: 78 (DNA)/SEQ ID NO: 93 (protein).MOR06185 represents an antibody having a variable heavy regioncorresponding to SEQ ID NO: 79 (DNA)/SEQ ID NO: 94 (protein). MOR06186represents an antibody having a variable heavy region corresponding toSEQ ID NO: 80 (DNA)/SEQ ID NO: 95 (protein). MOR06187 represents anantibody having a variable heavy region corresponding to SEQ ID NO: 81(DNA)/SEQ ID NO: 96 (protein). MOR06188 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 82 (DNA)/SEQ ID NO:97. MOR06189 represents an antibody having a variable heavy regioncorresponding to SEQ ID NO: 83 (DNA)/SEQ ID NO:98 (protein). MOR06190represents an antibody having a variable heavy region corresponding toSEQ ID NO: 84 (DNA)/SEQ ID NO: 99 (protein). MOR06192 represents anantibody having a variable heavy region corresponding to SEQ ID NO: 85(DNA)/SEQ ID NO: 100 (protein). MOR06195 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 86 (DNA)/SEQ ID NO:101 (protein). MOR06197 represents an antibody having a variable heavyregion corresponding to SEQ ID NO: 87 (DNA)/SEQ ID NO: 102 (protein).MOR06200 represents an antibody having a variable heavy regioncorresponding to SEQ ID NO: 88 (DNA)/SEQ ID NO: 103 (protein). MOR06201represents an antibody having a variable heavy region corresponding toSEQ ID NO: 89 (DNA)/SEQ ID NO: 104 (protein). MOR 06204 represents anantibody having a variable heavy region corresponding to SEQ ID NO: 90(DNA)/SEQ ID NO: 105 (protein). MOR06214 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 91 (DNA)/SEQ ID NO:106 (protein). MOR06278 represents an antibody having a variable lightregion corresponding to SEQ ID NO: 107 (DNA)/SEQ ID NO: 109 (protein).MOR 06279 represents an antibody having a variable light regioncorresponding to SEQ ID NO: 108 (DNA)/SEQ ID NO: 110 (protein).

Antibodies of the invention were characterized in Fab and/or IgG formatand comprise various combinations of the light and heavy chains ofoptimized and parental binders. FIG. 10 shows several non-limitingcombinations which can be used in connection with the present invention.

In one aspect, the invention provides methods for using antibodieshaving an antigen-binding region that can bind specifically to or has ahigh affinity for one or more regions of CD38, whose amino acid sequenceis depicted by SEQ ID NO: 71. An antibody is said to have a “highaffinity” for an antigen if the affinity measurement is at least 100 nM(monovalent affinity of Fab fragment). An antibody or antigen-bindingregion for use in the present invention preferably can bind to CD38 withan affinity of about less than 600 nM. Preferably, the antibody orantigen-binding region for use in the present invention can bind to CD38with an affinity of about less than 100 nM, more preferably less thanabout 60 nM, and still more preferably less than about 30 nM. Furtherpreferred are uses of antibodies that bind to CD38 with an affinity ofless than about 10 nM, and more preferably less than 3 about nM. Forinstance, the affinity of an antibody for use in the invention againstCD38 may be about 10.0 nM or 2.4 nM (monovalent affinity of Fabfragment).

Table 1 provides a summary of affinities of representative antibodies,as determined by surface plasmon resonance (Biacore) and FACS Scatchardanalysis:

TABLE 1 Antibody Affinities FACS Scatchard Antibody BIACORE (Fab)(IgG1)^(b) (Fab or IgG1) K_(D) [nM]^(a) K_(D) [nM] MOR03076 440/596 n.d.MOR03078 n.d. n.d. MOR03081 416/450 2.5 MOR03085 122 10 MOR03086 30 n.d.MOR03087 17/38 5 MOR03088 95 n.d. MOR03089 340 n.d. MOR03101 314 n.d.MOR03102 64 5 MOR03127 168 (54)^(c) n.d. MOR03128 117/84^(d)  n.d.MOR03129 43 n.d. MOR03130 n.d. n.d. MOR03131 451 n.d. Chimeric OKT10n.d. 8.28 ^(a)Fab format; analysis on human CD38 Fc-fusion aa 45-300^(b)IgG1 format; analysis with Raji cells ^(c)standard deviation (n = 3)^(d)standard deviation (n = 4)

With reference to Table 1, the affinity of LACs was measured by surfaceplasmon resonance (Biacore) on human CD38 Fc-fusion and by a flowcytometry procedure utilizing the CD38-expressing human Raji cell line.The Biacore studies were performed on directly immobilized antigen(CD38-Fc fusion protein). The Fab format of LACs exhibit an monovalentaffinity range between about 30 and 596 nM on immobilized CD38-Fc fusionprotein.

The IgG1 format was used for the cell-based affinity determination (FACSScatchard). The right column of Table 1 denotes the binding strength ofthe LACS in this format.

Another preferred feature of preferred antibodies for use in theinvention is their specificity for an area within the N-terminal regionof CD38. For example, LACs of the invention can bind specifically to theN-terminal region of CD38.

Optimized antibodies of the present invention were further characterizedas shown in Tables 2 and 3. Summaries are provided of affinities asdetermined by surface plasmon resonance (Biacore) and FACS Scatchardanalysis. Additionally, FACS-binding to human erythrocytes and ELISAbinding studies to CD38 Fc-Fusion protein have also been determined. Thecharacterizations show that several optimized binders show a reducedbinding to human erythrocytes and a higher ELISA signal as compared tothe parental clone. In addition derivatives of MOR03088 have an improvedaffinity as shown by FACS Scatchards and affinity determinations.

TABLE 2 Overview characterizations of affinity-matured clones: FACSanal. FACS-Binding ELISA Affinities to human CD38 Fc-Fusion KDs KDsScatchards [EC₅₀s] Erythrocytes^(b) protein^(b) (Biacore)^(a)(Bioveris)^(a) RPMI8226^(a) OPM2^(b) (Compared to Efficacy (% Reactivityof MOR# Optimization [nM] [pM] [nM] [nM] MOR03087) ADCC^(b,c) MOR03087)03087 Parental 5.68 48.77 5.37 17.4*/5.7 =MOR03087 + 100 6183 H-CDR213.47 25.98 28.06 8.91 <MOR03087 + 106 6184 H-CDR2 9.68 66.22 4.01 10.58~MOR03087 n.d. 150 6185 H-CDR2 4.39 13.69 7.30 11.50 <MOR03087 + 1426186 H-CDR2 4.62 5.09 6.47 15.57 <MOR03087 n.d. 117 6187 H-CDR2 12.4645.38 16.85 9.37 ~MOR03087 n.d. 145 6188 H-CDR2 3.98 59.32 22.71 20.15<MOR03087 n.d. 140 6189 H-CDR2 4.95 24.69 9.41 n.e. ~MOR03087 n.d. 1266190 H-CDR2 15.65 48.85 11.66 n.e. <MOR03087 n.d. 125 6192 H-CDR2 5.0174.73 7.07 n.e. ~MOR03087 n.d. 111 6195 H-CDR2 4.52 55.73 5.60 n.e.~MOR03087 n.d. 165 6197 H-CDR2 4.81 12.74 6.92 n.e. <MOR03087 n.d. 1386200 H-CDR2 7.92 59.91 5.02 7.15 >MOR03087 n.d. 144 6201 H-CDR2 6.8118.59 9.00 n.e. ~MOR03087 n.d. 137 03088 Parental 41.40 2149.92 24.6*15.3* no binding + 18 6204 H-CDR2 22.72 58.51 6.36 n.e. <MOR03087 n.d.56 6214 H-CDR2 5.26 93.65 5.32 n.e. <MOR03087 n.d. 109 6347 L-CDR3 n.d.n.d. n.d. n.d. n.d. n.d. n.d. 6348 L-CDR3 n.d. n.d. n.d. n.d. n.d. n.d.n.d. ^(a)Fab-format ^(b)IgG-format ^(c)Human Effector cells & RPMI8226Target cells (E:T ratio = 30:1) +: Killing of RPMI8226 cells in ADCCn.d.: not determined *different experiment

TABLE 3 EC₅₀ in FACS-Scatchard, ADCC and CDC CharacterizationsFACS-Scatchard ADCC CDC RPMI8226 CCRF-CEM OPM2 RPMI8226 CHO Anti-CD38EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ MAbs: [nM]^(a) [nM]^(a) [nM]^(b) [nM]^(b)[nM]^(a) MOR03087 6.3 14.7 17.54 0.14 3.4 MOR03088 24.6 25.5 2.6 n.e.n.e. MOR03080 1.8 2.6 1.9 0.13 1.9 ^(a)single measurement ^(b)mean from2 measurements

The type of epitope to which an antibody for use in the invention bindsmay be linear (i.e. one consecutive stretch of amino acids) orconformational (i.e. multiple stretches of amino acids). In order todetermine whether the epitope of a particular antibody is linear orconformational, the skilled worker can analyze the binding of antibodiesto overlapping peptides (e.g., 13-mer peptides with an overlap of 11amino acids) covering different domains of CD38. LACS may recognizediscontinuous or linear epitopes in the N-terminal region of CD38.Combined with the knowledge provided herein, the skilled worker in theart will know how to use one or more isolated epitopes of CD38 forgenerating antibodies having an antigen-binding region that is specificfor said epitopes (e.g. using synthetic peptides of epitopes of CD38 orcells expressing epitopes of CD38).

An antibody for use in the invention preferably is speciescross-reactive with humans and at least one other non-human species. Thenon-human species can be non-human primate, e.g. rhesus, baboon and/orcynomolgus. Other non-human species can be minipig, rabbit, mouse, ratand/or hamster. An antibody that is cross reactive with at least oneother species beside human can provide greater flexibility and benefitsover known anti-CD38 antibodies, for purposes of conducting in vivostudies in multiple species with the same antibody. An antibody that iscross reactive with minipig and/or rabbit, for example, can be acandidate for toxicology and safety studies.

Preferably, an antibody for use in the invention not only is able tobind to CD38, but also is able to mediate killing of a cell expressingCD38. More specifically, an antibody for use in the invention canmediate its therapeutic effect by depleting CD38-positive (e.g.,malignant) cells via antibody-effector functions. These functionsinclude antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC).

CD38-expression, however, is not only found on immune cells within themyeloid (e.g. monocytes, granulocytes) and lymphoid lineage (e.g.activated B and T-cells; plasma cells), but also on the respectiveprecursor cells. Since it is important that those cells are not affectedby antibody-mediated killing of malignant cells, the antibodies of thepresent invention are preferably not cytotoxic to precursor cells.

In addition to its catalytic activities as a cyclic ADP-ribose cyclaseand hydrolase, CD38 displays the ability to transduce signals ofbiological relevance (Hoshino et al., 1997; Ausiello et al., 2000).Those functions can be induced in vivo by, e.g. receptor-ligandinteractions or by cross-linking with agonistic anti-CD38 antibodies,leading, e.g. to calcium mobilization, lymphocyte proliferation andrelease of cytokines. Preferably, the antibodies of the presentinvention are non-agonistic antibodies.

Peptide Variants

Antibodies for use in the invention are not limited to the specificpeptide sequences provided herein. Rather, the invention also embodiesthe use of variants of these polypeptides. With reference to the instantdisclosure and conventionally available technologies and references, theskilled worker will be able to prepare, test and utilize functionalvariants of the antibodies disclosed herein, while appreciating thatvariants having the ability to mediate killing of a CD38+ target cellfall within the scope of the present invention. As used in this context,“ability to mediate killing of a CD38+ target cell” means a functionalcharacteristic ascribed to an anti-CD38 antibody for use in theinvention. Ability to mediate killing of a CD38+ target cell, thus,includes the ability to mediate killing of a CD38+ target cell, e.g. byADCC and/or CDC, or by toxin constructs conjugated to an antibody foruse in the invention.

A variant can include, for example, an antibody that has at least onealtered complementarity determining region (CDR) (hyper-variable) and/orframework (FR) (variable) domain/position, vis-à-vis a peptide sequencedisclosed herein. To better illustrate this concept, a brief descriptionof antibody structure follows.

An antibody is composed of two peptide chains, each containing one(light chain) or three (heavy chain) constant domains and a variableregion (VL, VH), the latter of which is in each case made up of four FRregions and three interspaced CDRs. The antigen-binding site is formedby one or more CDRs, yet the FR regions provide the structural frameworkfor the CDRs and, hence, play an important role in antigen binding. Byaltering one or more amino acid residues in a CDR or FR region, theskilled worker routinely can generate mutated or diversified antibodysequences, which can be screened against the antigen, for new orimproved properties, for example.

FIGS. 14 a (VH) and 14 b (VL) delineate the CDR and FR regions forcertain antibodies for use in the invention and compare amino acids at agiven position to each other and to corresponding consensus or “mastergene” sequences (as described in U.S. Pat. No. 6,300,064).

The skilled worker will be able to design peptide variants, the use ofwhich is within the scope of the present invention. It is preferred thatvariants are constructed by changing amino acids within one or more CDRregions; a variant might also have one or more altered frameworkregions. Alterations also may be made in the framework regions. Forexample, a peptide FR domain might be altered where there is a deviationin a residue compared to a germline sequence.

Furthermore, variants may be obtained by using one LAC as starting pointfor optimization by diversifying one or more amino acid residues in theLAC, preferably amino acid residues in one or more CDRs, and byscreening the resulting collection of antibody variants for variantswith improved properties. Particularly preferred is diversification ofone or more amino acid residues in CDR-3 of VL, CDR-3 of VH, CDR-1 of VLand/or CDR-2 of VH. Diversification can be done by synthesizing acollection of DNA molecules using trinucleotide mutagenesis (TRIM)technology (Virnekäs, B., Ge, L., Plückthun, A., Schneider, K. C.,Wellnhofer, G., and Moroney S. E. (1994) Trinucleotide phosphoramidites:ideal reagents for the synthesis of mixed oligonucleotides for randommutagenesis. Nucl. Acids Res. 22, 5600).

Conservative Amino Acid Variants

Polypeptide variants may be made that conserve the overall molecularstructure of an antibody peptide sequence described herein. Given theproperties of the individual amino acids, some rational substitutionswill be recognized by the skilled worker. Amino acid substitutions,i.e., “conservative substitutions,” may be made, for instance, on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.

For example, (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt α-helices. Similarly, certainamino acids, such as alanine, cysteine, leucine, methionine, glutamicacid, glutamine, histidine and lysine are more commonly found inα-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophanand threonine are more commonly found in β-pleated sheets. Glycine,serine, aspartic acid, asparagine, and proline are commonly found inturns. Some preferred substitutions may be made among the followinggroups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given theknown genetic code, and recombinant and synthetic DNA techniques, theskilled scientist readily can construct DNAs encoding the conservativeamino acid variants. In one particular example, amino acid position 3 inSEQ ID NOS: 5, 6, 7, and/or 8 can be changed from a Q to an E.

As used herein, “sequence identity” between two polypeptide sequencesindicates the percentage of amino acids that are identical between thesequences. “Sequence similarity” indicates the percentage of amino acidsthat either are identical or that represent conservative amino acidsubstitutions. Preferred polypeptide sequences of the invention have asequence identity in the CDR regions of at least 60%, more preferably,at least 70% or 80%, still more preferably at least 90% and mostpreferably at least 95%. Preferred antibodies also have a sequencesimilarity in the CDR regions of at least 80%, more preferably 90% andmost preferably 95%. Preferred polypeptide sequences of the inventionhave a sequence identity in the variable regions of at least 60%, morepreferably, at least 70% or 80%, still more preferably at least 90% andmost preferably at least 95%. Preferred antibodies also have a sequencesimilarity in the variable regions of at least 80%, more preferably 90%and most preferably 95%.

DNA Molecules of the Invention

The present invention also relates to uses of DNA molecules that encodean antibody for use in the invention. These sequences include, but arenot limited to, those DNA molecules set forth in FIGS. 1a and 2 a.

DNA molecules of the invention are not limited to the sequencesdisclosed herein, but also include variants thereof. DNA variants withinthe invention may be described by reference to their physical propertiesin hybridization. The skilled worker will recognize that DNA can be usedto identify its complement and, since DNA is double stranded, itsequivalent or homolog, using nucleic acid hybridization techniques. Italso will be recognized that hybridization can occur with less than 100%complementarity. However, given appropriate choice of conditions,hybridization techniques can be used to differentiate among DNAsequences based on their structural relatedness to a particular probe.For guidance regarding such conditions see, Sambrook et al., 1989(Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning:A laboratory manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, USA) and Ausubel et al., 1995 (Ausubel, F. M., Brent, R.,Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K.eds. (1995). Current Protocols in Molecular Biology. New York: JohnWiley and Sons).

Structural similarity between two polynucleotide sequences can beexpressed as a function of “stringency” of the conditions under whichthe two sequences will hybridize with one another. As used herein, theterm “stringency” refers to the extent that the conditions disfavorhybridization. Stringent conditions strongly disfavor hybridization, andonly the most structurally related molecules will hybridize to oneanother under such conditions. Conversely, non-stringent conditionsfavor hybridization of molecules displaying a lesser degree ofstructural relatedness. Hybridization stringency, therefore, directlycorrelates with the structural relationships of two nucleic acidsequences. The following relationships are useful in correlatinghybridization and relatedness (where T_(m) is the melting temperature ofa nucleic acid duplex):

-   -   a. T_(m)=69.3+0.41(G+C) %    -   b. The T_(m) of a duplex DNA decreases by 1° C. with every        increase of 1% in the number of mismatched base pairs.    -   c. (T_(m))_(μ2)−(T_(m))_(μ1)=18.5 log₁₀μ2/μ1        -   where μ1 and μ2 are the ionic strengths of two solutions.

Hybridization stringency is a function of many factors, includingoverall DNA concentration, ionic strength, temperature, probe size andthe presence of agents which disrupt hydrogen bonding. Factors promotinghybridization include high DNA concentrations, high ionic strengths, lowtemperatures, longer probe size and the absence of agents that disrupthydrogen bonding. Hybridization typically is performed in two phases:the “binding” phase and the “washing” phase.

First, in the binding phase, the probe is bound to the target underconditions favoring hybridization. Stringency is usually controlled atthis stage by altering the temperature. For high stringency, thetemperature is usually between 65° C. and 70° C., unless short (<20 nt)oligonucleotide probes are used. A representative hybridization solutioncomprises 6×SSC, 0.5% SDS, 5×Denhardt's solution and 100 μg ofnonspecific carrier DNA. See Ausubel et al., section 2.9, supplement 27(1994). Of course, many different, yet functionally equivalent, bufferconditions are known. Where the degree of relatedness is lower, a lowertemperature may be chosen. Low stringency binding temperatures arebetween about 25° C. and 40° C. Medium stringency is between at leastabout 40° C. to less than about 65° C. High stringency is at least about65° C.

Second, the excess probe is removed by washing. It is at this phase thatmore stringent conditions usually are applied. Hence, it is this“washing” stage that is most important in determining relatedness viahybridization. Washing solutions typically contain lower saltconcentrations. One exemplary medium stringency solution contains 2×SSCand 0.1% SDS. A high stringency wash solution contains the equivalent(in ionic strength) of less than about 0.2×SSC, with a preferredstringent solution containing about 0.1×SSC. The temperatures associatedwith various stringencies are the same as discussed above for “binding.”The washing solution also typically is replaced a number of times duringwashing. For example, typical high stringency washing conditionscomprise washing twice for 30 minutes at 55° C. and three times for 15minutes at 60° C.

Accordingly, the present invention includes the use of nucleic acidmolecules that hybridize to the molecules of set forth in FIGS. 1a and2a under high stringency binding and washing conditions, where suchnucleic molecules encode an antibody or functional fragment thereof foruses as described herein. Preferred molecules (from an mRNA perspective)are those that have at least 75% or 80% (preferably at least 85%, morepreferably at least 90% and most preferably at least 95%) homology orsequence identity with one of the DNA molecules described herein.

Functionally Equivalent Variants

Yet another class of DNA variants the use of which is within the scopeof the invention may be described with reference to the product theyencode (see the peptides listed in FIGS. 1b and 2b ). These functionallyequivalent genes are characterized by the fact that they encode the samepeptide sequences found in FIGS. 1b and 2b due to the degeneracy of thegenetic code.

It is recognized that variants of DNA molecules provided herein can beconstructed in several different ways. For example, they may beconstructed as completely synthetic DNAs. Methods of efficientlysynthesizing oligonucleotides in the range of 20 to about 150nucleotides are widely available. See Ausubel et al., section 2.11,Supplement 21 (1993). Overlapping oligonucleotides may be synthesizedand assembled in a fashion first reported by Khorana et al., J. Mol.Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section 8.2.Synthetic DNAs preferably are designed with convenient restriction sitesengineered at the 5′ and 3′ ends of the gene to facilitate cloning intoan appropriate vector.

As indicated, a method of generating variants is to start with one ofthe DNAs disclosed herein and then to conduct site-directed mutagenesis.See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typicalmethod, a target DNA is cloned into a single-stranded DNA bacteriophagevehicle. Single-stranded DNA is isolated and hybridized with anoligonucleotide containing the desired nucleotide alteration(s). Thecomplementary strand is synthesized and the double stranded phage isintroduced into a host. Some of the resulting progeny will contain thedesired mutant, which can be confirmed using DNA sequencing. Inaddition, various methods are available that increase the probabilitythat the progeny phage will be the desired mutant. These methods arewell known to those in the field and kits are commercially available forgenerating such mutants.

Recombinant DNA Constructs and Expression

The present invention further provides for the use of recombinant DNAconstructs comprising one or more of the nucleotide sequences of thepresent invention. The recombinant constructs are used in connectionwith a vector, such as a plasmid or viral vector, into which a DNAmolecule encoding an antibody for use in the invention is inserted.

The encoded gene may be produced by techniques described in Sambrook etal., 1989, and Ausubel et al., 1989. Alternatively, the DNA sequencesmay be chemically synthesized using, for example, synthesizers. See, forexample, the techniques described in OLIGONUCLEOTIDE SYNTHESIS (1984,Gait, ed., IRL Press, Oxford), which is incorporated by reference hereinin its entirety. Recombinant constructs of the invention are comprisedwith expression vectors that are capable of expressing the RNA and/orprotein products of the encoded DNA(s). The vector may further compriseregulatory sequences, including a promoter operably linked to the openreading frame (ORF). The vector may further comprise a selectable markersequence. Specific initiation and bacterial secretory signals also maybe required for efficient translation of inserted target gene codingsequences.

The present invention further provides for uses of host cells containingat least one of the DNAs disclosed herein. The host cell can bevirtually any cell for which expression vectors are available. It maybe, for example, a higher eukaryotic host cell, such as a mammaliancell, a lower eukaryotic host cell, such as a yeast cell, but preferablyis a prokaryotic cell, such as a bacterial cell. Introduction of therecombinant construct into the host cell can be effected by calciumphosphate transfection, DEAE, dextran mediated transfection,electroporation or phage infection.

Bacterial Expression

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and, if desirable, to provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus.

Bacterial vectors may be, for example, bacteriophage-, plasmid- orphagemid-based. These vectors can contain a selectable marker andbacterial origin of replication derived from commercially availableplasmids typically containing elements of the well known cloning vectorpBR322 (ATCC 37017). Following transformation of a suitable host strainand growth of the host strain to an appropriate cell density, theselected promoter is de-repressed/induced by appropriate means (e.g.,temperature shift or chemical induction) and cells are cultured for anadditional period. Cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the proteinbeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of antibodies or to screen peptidelibraries, for example, vectors which direct the expression of highlevels of fusion protein products that are readily purified may bedesirable.

Therapeutic Methods

Therapeutic methods involve administering to a subject in need oftreatment a therapeutically effective amount of an antibody contemplatedby the invention. A “therapeutically effective” amount hereby is definedas the amount of an antibody that is of sufficient quantity to depleteCD38-positive cells in a treated area of a subject—either as a singledose or according to a multiple dose regimen, alone or in combinationwith other agents, which leads to the alleviation of an adversecondition, yet which amount is toxicologically tolerable. The subjectmay be a human or non-human animal (e.g., rabbit, rat, mouse, monkey orother lower-order primate).

An antibody for use in the invention might be co-administered with knownmedicaments, and in some instances the antibody might itself bemodified. For example, an antibody could be conjugated to an immunotoxinor radioisotope to potentially further increase efficacy.

Disorders and conditions particularly suitable for treatment with anantibody are multiple myeloma (MM) and other haematological diseases,such as chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia(CML), acute myelogenous leukemia (AML), and acute lymphocytic leukemia(ALL). An antibody also might be used to treat inflammatory disease suchas rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).

To treat any of the foregoing disorders, pharmaceutical compositions foruse in accordance with the present invention may be formulated in aconventional manner using one or more physiologically acceptablecarriers or excipients. An antibody for use in the invention can beadministered by any suitable means, which can vary, depending on thetype of disorder being treated. Possible administration routes includeparenteral (e.g., intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous), intrapulmonary and intranasal, and,if desired for local immunosuppressive treatment, intralesionaladministration. In addition, an antibody for use in the invention mightbe administered by pulse infusion, with, e.g., declining doses of theantibody. Preferably, the dosing is given by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic. The amount to be administered willdepend on a variety of factors such as the clinical symptoms, weight ofthe individual, whether other drugs are administered. The skilledartisan will recognize that the route of administration will varydepending on the disorder or condition to be treated.

Determining a therapeutically effective amount of the novel polypeptide,according to this invention, largely will depend on particular patientcharacteristics, route of administration, and the nature of the disorderbeing treated. General guidance can be found, for example, in thepublications of the International Conference on Harmonisation and inREMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528(18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990).More specifically, determining a therapeutically effective amount willdepend on such factors as toxicity and efficacy of the medicament.Toxicity may be determined using methods well known in the art and foundin the foregoing references. Efficacy may be determined utilizing thesame guidance in conjunction with the methods described below in theExamples.

Diagnostic Methods

CD38 is highly expressed on hematological cells in certain malignancies;thus, an anti-CD38 antibody for use in the invention may be employed inorder to image or visualize a site of possible accumulation of malignantcells in a patient. In this regard, an antibody can be detectablylabeled, through the use of radioisotopes, affinity labels (such asbiotin, avidin, etc.) fluorescent labels, paramagnetic atoms, etc.Procedures for accomplishing such labeling are well known to the art.Clinical application of antibodies in diagnostic imaging are reviewed byGrossman, H. B., Urol. Clin. North Amer. 13:465-474 (1986)), Unger, E.C. et al., Invest. Radiol. 20:693-700 (1985)), and Khaw, B. A. et al.,Science 209:295-297 (1980)). Preferred antibodies or antigen-bindingregions of the invention for use as a diagnostic compound comprise avariable heavy chain sequence selected from the group consisting of SEQID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 and 106 and/ora variable light chain sequence selected from the group consisting ofSEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,109 and 110.

The detection of foci of such detectably labeled antibodies might beindicative of a site of tumor development, for example. In oneembodiment, this examination is done by removing samples of tissue orblood and incubating such samples in the presence of the detectablylabeled antibodies. In a preferred embodiment, this technique is done ina non-invasive manner through the use of magnetic imaging, fluorography,etc. Such a diagnostic test may be employed in monitoring the success oftreatment of diseases, where presence or absence of CD38-positive cellsis a relevant indicator. The invention also contemplates the use of ananti-CD38 antibody, as described herein for diagnostics in an ex vivosetting.

Therapeutic and Diagnostic Compositions

The antibodies for use in the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, wherein an antibody for use in the invention (includingany functional fragment thereof) is combined in a mixture with apharmaceutically acceptable carrier vehicle. Suitable vehicles and theirformulation are described, for example, in REMINGTON'S PHARMACEUTICALSCIENCES (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co.,1990). In order to form a pharmaceutically acceptable compositionsuitable for effective administration, such compositions will contain aneffective amount of one or more of the antibodies for use in the presentinvention, together with a suitable amount of carrier vehicle. Preferredantibodies or antigen-binding regions of the invention for use as adiagnostic compound comprise a variable heavy chain sequence selectedfrom the group consisting of SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105 and 106 and/or a variable light chain sequenceselected from the group consisting of SEQ ID NO: 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 109 and 110.

Preparations may be suitably formulated to give controlled-release ofthe active compound. Controlled-release preparations may be achievedthrough the use of polymers to complex or absorb anti-CD38 antibody. Thecontrolled delivery may be exercised by selecting appropriatemacromolecules (for example polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinyl-acetate, methylcellulose,carboxymethylcellulose, or protamine, sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled release preparations is to incorporate anti-CD38antibody into particles of a polymeric material such as polyesters,polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetatecopolymers. Alternatively, instead of incorporating these agents intopolymeric particles, it is possible to entrap these materials inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatine-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences (1980).

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules, orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compositions may, if desired, be presented in a pack or dispenserdevice, which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The invention further is understood by reference to the followingworking examples, which are intended to illustrate and, hence, not limitthe invention.

EXAMPLES Cell-lines

The following cell-lines were obtained from the European Collection ofCell Cultures (ECACC), the German Collection of Microorganisms (DSMZ) orthe American Type Culture collection (ATCC): hybridoma cell lineproducing the CD38 mouse IgG1 monoclonal antibody OKT10 (ECACC,#87021903), Jurkat cells (DSMZ, ACC282), LP-1 (DSMZ, ACC41), RPMI8226(ATCC, CCL-155), HEK293 (ATCC, CRL-1573), CHO-K1 (ATCC, CRL-61), Raji(ATCC, CCL-86), and OPM2 (DSMZ, ACC50).

Cells and Culture-Conditions

All cells were cultured under standardized conditions at 37° C. and 5%CO₂ in a humidified incubator. The cell-lines LP-1, RPMI8226, Jurkat andRaji were cultured in RPMI1640 (Pan biotech GmbH, #PO4-16500)supplemented with 10% FCS (PAN biotech GmbH, #P30-3302), 50 U/mlpenicillin, 50 μg/ml streptomycin (Gibco, #15140-122) and 2 mM glutamine(Gibco, #25030-024) and, in case of Jurkat- and Raji-cells, additionally10 mM Hepes (Pan biotech GmbH, #P05-01100) and 1 mM sodium pyruvate (Panbiotech GmbH, # PO4-43100) had to be added.

CHO-K1 and HEK293 were grown in DMEM (Gibco, #10938-025) supplementedwith 2 mM glutamine and 10% FCS. Stable CD38 CHO-K1 transfectants weremaintained in the presence of G418 (PAA GmbH, P11-012) whereas forHEK293 the addition of 1 mM sodium-pyruvate was essential. Aftertransient transfection of HEK293 the 10% FCS was replaced by Ultra lowIgG FCS (Invitrogen, #16250-078). The cell-line OKT10 was cultured inIDMEM (Gibco, #31980-022), supplemented with 2 mM glutamine and 20% FCS.

Preparation of Single Cell Suspensions from Peripheral Blood

All blood samples were taken after informed consent. Peripheral bloodmononuclear cells (PBMC) were isolated by Histopaque®-1077 (Sigma)according to the manufacturer's instructions from healthy donors. Redblood cells were depleted from these cell suspensions by incubation inACK Lysis Buffer (0.15 M NH4Cl, 10 mM KHCO₃, 0.1 M EDTA) for 5 min at RTor a commercial derivative (Bioscience, #00-4333). Cells were washedtwice with PBS and then further processed for flow cytometry or ADCC(see below).

Flow Cytometry (“FACS”)

All stainings were performed in round bottom 96-well culture plates(Nalge Nunc) with 2×10⁵ cells per well. Cells were incubated with Fab orIgG antibodies at the indicated concentrations in 50 μl FACS buffer(PBS, 3% FCS, 0.02% NaN₃) for 40 min at 4° C. Cells were washed twiceand then incubated with R-Phycoerythrin (PE) conjugated goat-anti-humanor goat-anti-mouse IgG (H+L) F(ab′)₂ (Jackson Immuno Research), diluted1:200 in FACS buffer, for 30 min at 4° C. Cells were again washed,resuspended in 0.3 ml FACS buffer and then analyzed by flow cytometry ina FACSCalibur (Becton Dickinson, San Diego, Calif.).

For FACS based Scatchard analyses RPMI8226 cells were stained with at 12different dilutions (1:2^(n)) starting at 12.5 μg/ml (IgG) finalconcentration. At least two independent measurements were used for eachconcentration and K_(D) values extrapolated from median fluorescenceintensities according to Chamow et al. (1994).

Surface Plasmon Resonance

The kinetic constants k_(on) and k_(off) were determined with serialdilutions of the respective Fab binding to covalently immobilizedCD38-Fc fusion protein using the BIAcore 3000 instrument (Biacore,Uppsala, Sweden). For covalent antigen immobilization standard EDC-NHSamine coupling chemistry was used. For direct coupling of CD38 Fc-fusionprotein CM5 sensor chips (Biacore) were coated with ˜600-700 RU in 10 mMacetate buffer, pH 4.5. For the reference flow cell a respective amountof HSA (human serum albumin) was used. Kinetic measurements were done inPBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄ pH 7.4) at aflow rate of 20 μl/min using Fab concentration range from 1.5-500 nM.Injection time for each concentration was 1 min, followed by 2 mindissociation phase. For regeneration 5 μl 10 mM HCl was used. Allsensograms were fitted locally using BIA evaluation software 3.1(Biacore).

Example 1 Antibody Generation from HuCAL Libraries

For the generation of therapeutic antibodies against CD38, selectionswith the MorphoSys HuCAL GOLD® phage display library were carried out.HuCAL GOLD® is a Fab library based on the HuCAL® concept (Knappik etal., 2000; Krebs et al., 2001), in which all six CDRs are diversified,and which employs the CysDisplay™ technology for linking Fab fragmentsto the phage surface (Löhning, 2001).

A. Phagemid rescue, phage amplification and purification

HuCAL GOLD® phagemid library was amplified in 2×TY medium containing 34μg/ml chloramphenicol and 1% glucose (2×TY-CG). After helper phageinfection (VCSM13) at an OD600 of 0.5 (30 min at 37° C. without shaking;30 min at 37° C. shaking at 250 rpm), cells were spun down (4120 g; 5min; 4° C.), resuspended in 2×TY/34 μg/ml chloramphenicol/50 μg/mlkanamycin and grown overnight at 22° C. Phages were PEG-precipitatedfrom the supernatant, resuspended in PBS/20% glycerol and stored at −80°C. Phage amplification between two panning rounds was conducted asfollows: mid-log phase TG1 cells were infected with eluted phages andplated onto LB-agar supplemented with 1% of glucose and 34 μg/ml ofchloramphenicol (LB-CG). After overnight incubation at 30° C., colonieswere scraped off, adjusted to an OD600 of 0.5 and helper phage added asdescribed above.

B. Pannings with HuCAL GOLD®

For the selections HuCAL GOLD® antibody-phages were divided into threepools corresponding to different VH master genes (pool 1: VH1/5λκ, pool2: VH3 λκ, pool 3: VH2/4/6 λκ). These pools were individually subjectedto 3 rounds of whole cell panning on CD38-expressing CHO-K1 cellsfollowed by pH-elution and a post-adsorption step on CD38-negativeCHO-K1-cells for depletion of irrelevant antibody-phages. Finally, theremaining antibody phages were used to infect E. coli TG1 cells. Aftercentrifugation the bacterial pellet was resuspended in 2×TY medium,plated on agar plates and incubated overnight at 30° C. The selectedclones were then scraped from the plates, phages were rescued andamplified. The second and the third round of selections were performedas the initial one.

The Fab encoding inserts of the selected HuCAL GOLD® phages weresubcloned into the expression vector pMORPH®x9_Fab_FS (Rauchenberger etal., 2003) to facilitate rapid expression of soluble Fab. The DNA of theselected clones was digested with XbaI and EcoRI thereby cutting out theFab encoding insert (ompA-VLCL and phoA-Fd), and cloned into theXbaI/EcoRI cut vector pMORPH®x9_Fab_FS. Fab expressed in this vectorcarry two C-terminal tags (FLAG™ and Strep-tag® II) for detection andpurification.

Example 2 Biological Assays

Antibody dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity was measured according to a published protocol based onflow-cytometry analysis (Naundorf et al., 2002) as follows:

ADCC:

For ADCC measurements, target cells (T) were adjusted to 2.0E+05cells/ml and labeled with 100 ng/ml Calcein AM (Molecular Probes,C-3099) in RPMI1640 medium (Pan biotech GmbH) for 2 minutes at roomtemperature. Residual calcein was removed by 3 washing steps in RPMI1640medium. In parallel PBMC were prepared as source for (natural killer)effector cells (E), adjusted to 1.0E+07 and mixed with the labeledtarget cells to yield a final E:T-ratio of 50:1 or less, depending onthe assay conditions. Cells were washed once and the cell-mixresuspended in 200 μl RPMI1640 medium containing the respective antibodyat different dilutions. The plate was incubated for 4 hrs understandardized conditions at 37° C. and 5% CO₂ in a humidified incubator.Prior to FACS analysis cells were labeled with propidium-iodide (PI) andanalyzed by flow-cytometry (Becton-Dickinson). Between 50.000 and150.000 events were counted for each assay.

The following equation gave rise to the killing activity [in %]:

$\frac{{ED}^{A}}{{EL}^{A} + {ED}^{A}} \times 100$

with ED^(A)=events dead cells (calcein+PI stained cells), andEL^(A)=events living cells (calcein stained cells)

CDC:

For CDC measurements, 5.0E+04 CD38 CHO-K1 transfectants were added to amicrotiter well plate (Nunc) together with a 1:4 dilution of human serum(Sigma, #S-1764) and the respective antibody. All reagents and cellswere diluted in RPMI1640 medium (Pan biotech GmbH) supplemented with 10%FCS. The reaction-mix was incubated for 2 hrs under standardizedconditions at 37° C. and 5% CO₂ in a humidified incubator. As negativecontrols served either heat-inactivated complement or CD38-transfectantswithout antibody. Cells were labeled with PI and subjected toFACS-analysis.

In total 5000 events were counted and the number of dead cells atdifferent antibody concentrations used for the determination of EC50values. The following equation gave rise to the killing activity [in %]:

$\frac{{ED}^{C}}{{EL}^{C} + {ED}^{C}} \times 100$

with ED^(C)=events dead cells (PI stained cells), andEL^(C)=events living cells (unstained)

Cytotoxicity values from a total of 12 different antibody-dilutions(1:2^(n)) in triplicates were used in ADCC and duplicates in CDC foreach antibody in order obtain EC-50 values with a standard analysissoftware (PRISM®, Graph Pad Software).

Example 3 Generation of Stable CD38-Transfectants and CD38 Fc-FusionProteins

In order to generate CD38 protein for panning and screening twodifferent expression systems had to be established. The first strategyincluded the generation of CD38-Fc-fusion protein, which was purifiedfrom supernatants after transient transfection of HEK293 cells. Thesecond strategy involved the generation of a stable CHO-K1-cell line forhigh CD38 surface expression to be used for selection of antibody-phagesvia whole cell panning.

As an initial step Jurkat cells (DSMZ ACC282) were used for thegeneration of cDNA (Invitrogen) followed by amplification of the entireCD38-coding sequence using primers complementary to the first 7 and thelast 9 codons of CD38, respectively (primer MTE001 & MTE002rev; Table4). Sequence analysis of the CD38-insert confirmed the published aminoacid sequence by Jackson et al. (1990) except for position 49 whichrevealed a glutamine instead of a tyrosine as described by Nata et al.(1997). For introduction of restriction endonuclease sites and cloninginto different derivatives of expression vector pcDNA3.1 (Stratagene),the purified PCR-product served as a template for the re-amplificationof the entire gene (primers MTE006 & MTE007rev, Table 4) or a part(primers MTE004 & MTE009rev, Table 4) of it. In the latter case afragment encoding for the extracellular domain (aa 45 to 300) wasamplified and cloned in frame between a human Vkappa leader sequence anda human Fc-gamma 1 sequence. This vector served as expression vector forthe generation of soluble CD38-Fc fusion-protein. AnotherpcDNA3.1-derivative without leader-sequence was used for insertion ofthe CD38 full-length gene. In this case a stop codon in front of theFc-coding region and the missing leader-sequence gave rise toCD38-surface expression. HEK293 cells were transiently transfected withthe Fc-fusion protein vector for generation of soluble CD38 Fc-fusionprotein and, in case of the full-length derivative, CHO-K1-cells weretransfected for the generation of a stable CD38-expressing cell line.

TABLE 4 Primer # Sequence (5′→3′) MTE001 ATG GCC AAC TGC GAG TTC AGC(SEQ ID NO: 123) MTE002rev TCA GAT CTC AGA TGT GCA AGA TGA ATC(SEQ ID NO: 124) MTE004 TT GGT ACC AGG TGG CGC CAG CAG TG(SEQ ID NO: 125) MTE006 TT GGT ACC ATG GCC AAC TGC GAG (SEQ ID NO: 126)MTE007rev CCG ATA TCA* GAT CTC AGA TGT GCA AGA TG (SEQ ID NO: 127)MTE009rev CCG ATA TC   GAT CTC AGA TGT GCA AGA TG (SEQ ID NO: 128)*leading to a stop codon (TGA) in the sense orientation.

Example 4 Cloning, Expression and Purification of HuCAL® IgG1

In order to express full length IgG, variable domain fragments of heavy(VH) and light chains (VL) were subcloned from Fab expression vectorsinto appropriate pMORPH®_hIg vectors (see FIGS. 7 to 9). Restrictionendonuclease pairs BlpI/MfeI (insert-preparation) and BlpI/EcoRI(vector-preparation) were used for subcloning of the VH domain fragmentinto pMORPH®_hIgG1. Enzyme-pairs EcoRV/HpaI (lambda-insert) andEcoRV/BsiWI (kappa-insert) were used for subcloning of the VL domainfragment into the respective pMORPH®_hIgκ_1 or pMORPH®_h_Igλ_1 vectors.Resulting IgG constructs were expressed in HEK293 cells (ATCC CRL-1573)by transient transfection using standard calcium phosphate—DNAcoprecipitation technique.

IgGs were purified from cell culture supernatants by affinitychromatography via Protein A Sepharose column. Further down streamprocessing included a buffer exchange by gel filtration and sterilefiltration of purified IgG. Quality control revealed a purity of >90% byreducing SDS-PAGE and >90% monomeric IgG as determined by analyticalsize exclusion chromatography. The endotoxin content of the material wasdetermined by a kinetic LAL based assay (Cambrex European EndotoxinTesting Service, Belgium).

Example 5 Generation and Production of Chimeric OKT10 (chOKT10; SEQ IDNOS: 72 and 73)

For the construction of chOKT10 the mouse VH and VL regions wereamplified by PCR using cDNA prepared from the murine OKT10 hybridomacell line (ECACC #87021903). A set of primers was used as published(Dattamajumdar et al., 1996; Zhou et al., 1994). PCR products were usedfor Topo-cloning (Invitrogen; pCRII-vector) and single coloniessubjected to sequence analysis (M13 reverse primer) which revealed twodifferent kappa light chain sequences and one heavy chain sequence.According to sequence alignments (EMBL-nucleotide sequence database) andliterature (Krebber et al, 1997) one of the kappa-sequence belongs tothe intrinsic repertoire of the tumor cell fusion partner X63Ag8.653 andhence does not belong to OKT10 antibody. Therefore, only the new kappasequence and the single VH-fragment was used for further cloning. Bothfragments were reamplified for the addition of restriction endonucleasesites followed by cloning into the respective pMORPH® IgG1-expressionvectors. The sequences for the heavy chain (SEQ ID NO: 72) and lightchain (SEQ ID NO: 73) are given in FIG. 6. HEK293 cells were transfectedtransiently and the supernatant analyzed in FACS for the chimeric OKT10antibody binding to the CD38 over-expressing Raji cell line (ATCC).

Example 6 Cross Reactivity Analysis by FACS (MOR 03087 and MOR 03088) 1.Materials and Methods:

FIGS. 11 and 12 show FACS analyses of lymphocytes and erythrocytes:EDTA-treated blood samples were obtained from healthy humans (afterobtaining informed consent) and from non human primates (Rhesus,Cynomolgus and Marmoset) and were subjected to density gradientcentrifugation using the Histopaque cell separation system according tothe instructions of the supplier (Sigma). For FACS-analysis, cells fromthe interphase (PBMC-fraction) and pellet (Erythrocyte-fraction) wereincubated with anti-CD38 HuCAL® antibodies in different formats

An overview of cross reactivity profiles of different anti CD38antibodies is shown in FIG. 13

2. Summary and Conclusion:

The results show that among all CD38 antibodies only MOR03087 andMOR03088 showed cross-reactivity to marmoset PBMCs. Surprisingly,CD38-expression on marmoset erythrocytes is almost not detectable ascompared to the strong expression on cynomolgus and rhesus erythrocytes.Thus, the CD38 expression on marmoset erythrocytes and PBMCs is morereflecting the human situation, where CD38 expression is low onerythrocytes and moderate to high on PBMCs. Marmoset is thereforeconsidered to be suited as a model to study toxicity of moleculesbinding to CD38.

Based on the above study, it was decided to further optimize the bindersMOR 03087 and MOR 03088, as described elsewhere in the specification,see e.g. paragraph relating to “Antibodies for use in the invention”. Aperson skilled in the art would expect that also the derivativeantibodies of the parentals would show a comparable cross reactivityprofile.

REFERENCES

-   Antonelli, A., Baj., G., Marchetti., P., Fallahi, P., Surico, N.,    Pupilli, C., Malavasi, F., Ferrannini, P. (2001). Human anti-CD38    autoantibodies raise intracellular calcium and stimulate insulin    release in human pancreatic islets. Diabetes 50: 985-991-   Ausiello C. M., Urbani F., Lande R., la Sala A., Di Carlo B., Baj    G., Surico N., Hilgers J., Deaglio S., Funaro A., Malavasi F. (2000)    Functional topography of discrete domains of human CD38. Tissue    Antigens. 2000 December; 56(6):539-47.-   Chamow, S. M., Zhang, D. Z., Tan, X. Y, Mathre, S. M., Marsters, S.    A., Peers, D. H., Byrn, R. A., Ashknazi, A., Junghans, R. P (1994).    humanized, bispecific immunoadhesin-antibody that retargets CD3+    effectors to kill HIV-1-infected cells. J Immunol. 1994 Nov. 1;    153(9):4268-80-   Dattamajumdar, A. K., Jacobsen, D. P., Hood, L. E., Osman, G. E.    (1996). Rapid cloning of rearranged mouse immunoglobulin variable    genes. Immunogentetics 43, 141-151-   Ellis J. H., Barber, K. A., Tuft, A., Hale, C., Lewis, A. P.,    Glennie, M. J., Stevenson, G. T., and Crowe, J. (1995). Engineered    anti-CD38 monoclonal antibodies for immunotherapy of multiple    myeloma. J. Immunol. 155:925-937.-   Ferrero, E., Orciani, M., Vacca, P., Ortolan, E., Crovella, S.,    Titti, F., Saccucci, F., Malavasi, F. (2004). Characterization and    phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus    macaque. BMC Immunology 5:21-   Flavell, D. J., Boehm, D. A., Noss, A., Warnes, S. L., and    Flavell, S. U. Therapy of human T-cell acute lymphoblastic leukaemia    with a combination of anti-CD7 and anti-CD38-saporin immunotoxins is    significantly better than therapy with each individual immunotoxin,    Br. J. Cancer. 84:571-578 (2001).-   Funaro, A., Spagnoli, G. C., Ausiello, C. M., Alessio, M., Roggero,    S., Delia, D., Zaccolo, M., and Malavasi, F. (1990) Involvement of    the multilineage CD38 molecule in a unique pathway of cell    activation and proliferation. J. Immunol. 145, 2390-2396.-   Golay, J., Zaffaroni, Luisella, Vaccari, T., Lazzari, M., Borleri,    G.-M., Bernasconi, S., Tedesco, F., Rambaldi, Al, Introna, M.    (2000). Biological response of B lymphoma to anti-CD20 monoclonal    antibody in vitro: CD55 and CD59 regulate complement-mediated cell    lysis. Blood 95: 3900-3908.-   Hayashi, T., Treon, S. P., Hideshima, T., Tai, Y-T., Akiyama, M.,    Richardson, R., Chauhan, D., Grewal, I. S., Anderson, K. C. (2003).    Recombinant humanized anti-CD40 monoclonal antibody triggers    autologous antibody-dependent cell-mediated cytotoxicity against    multiple myeloma. Br. J. Heamatol. 121, 592-596.-   Hoshino S., Kukimoto I., Kontani K., Inoue S., Kanda Y., Malavasi    F., Katada T. (1997) Mapping of the catalytic and epitopic sites of    human CD38/NAD+ glycohydrolase to a functional domain in the    carboxyl terminus. J Immunol. 158(2):741-7.-   Jackson D. G., Bell J. I. (1990) Isolation of a cDNA encoding the    human CD38 (T10) molecule, a cell surface glycoprotein with an    unusual discontinuous pattern of expression during lymphocyte    differentiation. J Immunol. 144(7):2811-5.-   Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M.,    Wellnhofer, G., Hoess, A., Wolle, J., Pluckthun, A., and    Virnekas, B. (2000). Fully synthetic human combinatorial antibody    libraries (HuCAL) based on modular consensus frameworks and CDRs    randomized with trinucleotides. J Mol Biol 296, 57-86.-   Kono, K., Takahashi, A., Ichihara, F., Sugai, H., Fujii, H., and    Matsumoto, Y. (2002). Impaired antibody-dependent cellular    cytotoxicity mediated by Herceptin in patients with gastritic    cancer. Cancer Res. 62, 5813-5817.-   Konopleva M., Estrov Z., Zhao S., Andreeff M., Mehta K. (1998)    Ligation of cell surface CD38 protein with agonistic monoclonal    antibody induces a cell growth signal in myeloid leukemia cells. J    Immunol. 161(9):4702-8.-   Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda,    J., Bossard, H. R., Plückthun, A. (1997). Reliable cloning of    functional antibody variable domains from hybridomas and spleen cell    repertoires employing a reengineered phage display system. J. Imm.    Meth. 201, 35-55.-   Krebs, B., Rauchenberger, R., Reiffert, S., Rothe, C., Tesar, M.,    Thomassen, E., Cao, M., Dreier, T., Fischer, D., Hoss, A., Inge, L.,    Knappik, A., Marget, M., Pack, P., Meng, X. Q., Schier, R.,    Sohlemann, P., Winter, J., Wolle, J., and Kretzschmar, T. (2001).    High-throughput generation and engineering of recombinant human    antibodies. J Immunol Methods 254, 67-84.-   Löhning, C. (2001). Novel methods for displaying    (poly)peptides/proteins on bacteriophage particles via disulfide    bonds. WO 01/05950.-   Malavasi, F., Caligaris-Cappio, F., Milanese, C., Dellabona, P.,    Richiardi, P., Carbonara, A. O. (1984). Characterization of a murine    monoclonal antibody specific for human early lymphohemopoietic    cells. Hum. Immunol. 9: 9-20-   Maloney, D. G., Smith, B., and Rose, A. (2002). Rituximab: Mechanism    of Action and Resistance. Sem. Oncol. 29, 2-9.-   Marchetti, P., Antonelli, A., Lupi, R., Marselli, L., Fallahi, P.,    Nesti, C., Baj, G., Ferrannini, E. (2002). Prolonged in vitro    exposure to autoantibodies against CD38 impairs the function and    survival of human pancreatic islets. Diabetes 51, 474-477.-   Mehta, K., Ocanas, L., Malavasi, f., Marks; J. W., Rosenblum, M. G    (2004). Retinoic acid-induced CD38 antigen as a target for    immunotoxin-mediated killing of leukemia cells. Mol. Cancer Ther. 3,    345-352-   Namba, M., Otsuki, T., Mori, M., Togawa, A., Wada, H., Sugihara, T.,    Yawata, Y., Kimoto, T. (1989). Establishment of five human myeloma    cell lines. In Vitro Cell Dev. Biol. 25: 723.-   Nata K., Takamura T., Karasawa T., Kumagai T., Hashioka W., Tohgo    A., Yonekura H., Takasawa S., Nakamura S., Okamoto H. (1997). Human    gene encoding CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose    hydrolase): organization, nucleotide sequence and alternative    splicing. Gene 186(2):285-92.-   Naundorf, S., Preithner, S., Mayer, P., Lippold, S., Wolf, A.,    Hanakam, F., Fichtner, I., Kufer, P., Raum, T., Riethmüller, G.,    Baeuerle, P. A., Dreier, T. (2002). Int. J. Cancer 100, 101-110.-   Plückthun A, and Pack P. (1997) New protein engineering approaches    to multivalent and bispecific antibody fragments. Immunotechnology    3(2):83-105.-   Rauchenberger R., Borges E., Thomassen-Wolf E., Rom E., Adar R.,    Yaniv Y., Malka M., Chumakov I., Kotzer S., Resnitzky D., Knappik    A., Reiffert S., Prassler J., Jury K., Waldherr D., Bauer S.,    Kretzschmar T., Yayon A., Rothe C. (2003). Human combinatorial Fab    library yielding specific and functional antibodies against the    human fibroblast growth factor receptor 3. J Biol Chem.    278(40):38194-205.-   Reff, M. E., Curler, K., Chambers, K. S., Chinn, P. C., Leonard, J.    E., Raab, R., Newman, R. A.,-   Hanna, N., Anderson, D. R. (1994). Depletion of B cells in vivo by a    chimeric mouse human monoclonal antibody to CD20. Blood 83: 435-445-   Santin, A. D., Bellone, S., Gokden, M., Palmieri, M., Dunn, D.,    Agha, J., Roman, J. J., Hutchins, L., Pecorelli, S., O'Brian, T.,    Cannon, M. J., Parham, G. P. (2002). Overexpression of HER-2/Neu in    Uterine serous papillary cancer. Cl. Cancer Res. 8: 1271-1279.-   Shinkawa, T., Nakamura, K., Yamane, N., Shoji-Hosaka, E., Kanda, Y.,    Sakurada, M., Uchida, K., Anazawa, H., Satoh, M., Yamasaki, M.,    Hanai, N., Shitara, K. (2003). The absence of fucose but Not the    presence of galactose or bisectin N-Acteylglucosamine of human IgG1    complex-type oligoscaccharides shows the critical role of enhancing    antibody-dependent cellular cytotoxicity. J. Biol. Chem. 278,    3466-3473.-   Zhou, H., Fisher, R. J., Papas, T. S. (1994). Optimization of primer    sequences for mouse scFv repertoire display library construction.    Nucleic Acids Res. 22: 888-889.

1-3. (canceled)
 4. An isolated antibody or functional fragment thereofthat is specific for CD38, which comprises (i) a variable heavy chaindepicted in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105or 106 or (ii) a variable heavy chain that has at least a sixty percentidentity to a variable heavy chain depicted in SEQ ID NO: 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105 or
 106. 5-14. (canceled)
 15. Anucleic acid sequence encoding a variable light chain of an isolatedantigen-binding region, which comprises (i) a sequence selected from thegroup consisting of SEQ ID NOS: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 107 and 108 or (ii) a nucleic acid sequence thathybridizes under high stringency conditions to the complementary strandof SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 107 or 108 wherein said antigen-binding region is specific for CD38.16. A vector comprising a nucleic acid sequence according to claim 15.17. An isolated cell comprising a vector according to claim
 16. 18-19.(canceled)
 20. A pharmaceutical composition comprising an antibody orfunction fragment thereof according to claim 4 and a pharmaceuticallyacceptable carrier or excipient therefor.
 21. A method for treating adisorder or condition associated with the undesired presence of CD38+cells, comprising administering to a subject in need thereof aneffective amount of the pharmaceutical composition according to claim20. 22-44. (canceled)
 45. A method of inducing specific killing of tumorcells that express CD38, comprising the step of contacting said cellswith a sufficient amount of an isolated human or humanized anti-CD38antibody or antigen-binding fragment thereof, which comprises the threevariable heavy chain complementarity determining regions (H-CDRs) andthe three variable light-chain complementarity determining regions(L-CDRs) that are in the variable heavy chain and variable light chainpairs selected from: (i) SEQ ID NO: 18 and 48, (ii) SEQ ID NO: 20 and50, (iii) SEQ ID NO: 21 and 51, (iv) SEQ ID NO: 22 and 52, and (v) SEQID NO: 25 and
 55. 46. The method according to claim 45, wherein theisolated antibody or antigen binding fragment thereof comprises thethree H-CDRs in SEQ ID NO: 18 and the three L-CDRs depicted in SEQ IDNO:
 48. 47. The method according to claim 45, wherein the isolatedantibody or antigen binding fragment thereof comprises the three H-CDRsin SEQ ID NO: 20 and the three L-CDRs depicted in SEQ ID NO:
 50. 48. Themethod according to claim 45, wherein the isolated antibody or antigenbinding fragment thereof comprises the three H-CDRs in SEQ ID NO: 21 andthe three L-CDRs depicted in SEQ ID NO:
 51. 49. The method according toclaim 45, wherein the isolated antibody or antigen binding fragmentthereof comprises the three H-CDRs in SEQ ID NO: 22 and the three L-CDRsdepicted in SEQ ID NO:
 52. 50. The method according to claim 45, whereinthe isolated antibody or antigen binding fragment thereof comprises thethree H-CDRs in SEQ ID NO: 25 and the three L-CDRs depicted in SEQ IDNO:
 55. 51. The method according to claim 45, wherein the isolatedantibody or antigen binding fragment thereof comprises a variable heavychain depicted in SEQ ID NO: 18, 20, 21, 22, or
 25. 52. The methodaccording to claim 45, wherein the isolated antibody or antigen bindingfragment thereof comprises a variable light chain depicted in SEQ ID NO:48, 50, 51, 52, or
 55. 53. The method according to claim 48, wherein theisolated antibody or antigen binding fragment thereof comprises avariable heavy chain at least 80% identical to that depicted in SEQ IDNO:
 21. 54. The method according to claim 53, wherein the isolatedantibody or antigen binding fragment thereof comprises a variable heavychain at least 90% identical to that depicted in SEQ ID NO:
 21. 55. Themethod according to claim 54, wherein the isolated antibody or antigenbinding fragment thereof comprises a variable heavy chain depicted inSEQ ID NO:
 21. 56. The method according to claim 51, wherein theisolated antibody or antigen binding fragment thereof comprises avariable heavy chain depicted in SEQ ID NO:
 22. 57. The method accordingto claim 48, wherein the isolated antibody or antigen binding fragmentthereof comprises a variable light chain at least 80% identical to thatdepicted in SEQ ID NO:
 51. 58. The method according to claim 57, whereinthe isolated antibody or antigen binding fragment thereof comprises avariable light chain at least 90% identical to that depicted in SEQ IDNO:
 51. 59. The method according to claim 58, wherein the isolatedantibody or antigen binding fragment thereof comprises a variable lightchain depicted in SEQ ID NO:
 51. 60. The method according to claim 51,wherein the isolated antibody or antigen binding fragment thereofcomprises a variable light chain depicted in SEQ ID NO:
 52. 61. Themethod according to claim 51, wherein the isolated antibody or antigenbinding fragment thereof comprises a heavy chain depicted in SEQ ID NO:18, and a light chain depicted in SEQ ID NO:
 48. 62. The methodaccording to claim 51, wherein the isolated antibody or antigen bindingfragment thereof comprises a heavy chain depicted in SEQ ID NO: 20, anda light chain depicted in SEQ ID NO:
 50. 63. The method according toclaim 48, wherein the isolated antibody or antigen binding fragmentthereof comprises a heavy chain depicted in SEQ ID NO: 21, and a lightchain depicted in SEQ ID NO:
 51. 64. The method according to claim 51,wherein the isolated antibody or antigen binding fragment thereofcomprises a heavy chain depicted in SEQ ID NO: 22 and a light chaindepicted in SEQ ID NO:
 52. 65. The method according to claim 51, whereinthe isolated antibody or antigen binding fragment thereof comprises aheavy chain depicted in SEQ ID NO: 25 and a light chain depicted in SEQID NO:
 55. 66. The method according to claim 45, wherein the isolatedantigen binding fragment is an scFv, Fab or F(ab′).sub.2 fragment. 67.The method according to claim 45, wherein the isolated antibody is anIgG.
 68. The method according to claim 67, wherein the isolated antibodyis an IgG1.